Question

You are responsible for characterizing a novel mutant version of PAH from a patient experiencing mild symptoms of the disease. You first task is to purify the enzyme responsible. You decide to generate a construct to express the protein in E.coli.

Step 3-1: PCR to amplify the mutated gene. You have the following samples: a. Genomic DNA from the patients hepatocytes. b. Genomic DNA from the blood cells. c. mRNA from the hepatocytes d. mRNA from the blood cells e. cDNA from the hepatocytes f. cDNA from the blood cells. Which would you choose as your template and why?

Step 3-2: Creating a construct. Which sequence elements would your DNA construct require for expression in E. coli to be successful?

Circle all that apply. Bacterial origin of

replication (ORI) Human ORI

Human TATA box sequence Explain:

Bacterial Ribosome Binding Site (RBS)
Human (RBS)

Antibiotic resistance gene

Bacterial Terminator sequence

Human DNA Polymerase binding site
T7 RNA polymerase binding site

         

Step 3-3: You complete the PCR and clone it into your DNA construct. You sequence the cloned gene and find four changes relative to your normal PAH sequence:

1. A mutation of a Tyr to a Ser that is observed in approximately 10% of human DNA sequences and localizes to the surface of the protein.

2. A mutation of a Leu to an Ala that is a novel mutation and localized to coiled-coil interaction between the tetrameric PAH subunits.

3. A mutation of a proline to an alanine that is a novel mutation and localized to a loop near the active site of the enzyme.

Circle the two out of the three mutations that are MOST likely to be a potential cause of the patient’s illness.

Explain your choice:

4. You know that the pI for this protein is ~ 6.2 based on sequence analysis. You decide to try purifying it using a anion exchange column.
Step 4-1: Choose a suitable buffer from the list below and explain your choice:

a. 30mMNaCl,100mMTrispH7.5
b. 30mMNaCl,100mMMes pH6.5 c. 30 mM NaCl, 100 mM Citrate pH 5.5

Buffer Choice: _________ Explain:

d. 300mMNaCl,100mMTrispH7.5
e. 300mMNaCl,100mMMes pH6.5 f. 300 mM NaCl, 100 mM Citrate pH 5.5

    

Step 2: You pass the E.coli lysate after expression over the anion exchange column and test the eluted fractions for the presence of iron. Finding no iron-containing fractions, you analyze the results by SDS-PAGE. Here is what you observe:

Circle yes or no to answer :

Was the PAH:

Expressed?

The correct size?

Soluble?

Bound by the column?

Present in the fractions?

Yes No

Yes No

Yes No

Yes No

Yes No

State a hypothesis that explains the observations above:

Briefly describe one experiment to test your hypothesis:

5. In order to determine which of the two mutations you identified in step 3-3 is responsible for the symptoms in the patient, what biochemical analyses would you perform using your purified protein? Briefly explain, stating the objective of the analyses, the technique/method you would use, the controls you would need and what outcome you would expect for each possibility.

Objective:

Methods/Techniques used:

Outcome/results expected (for each possibility):

6. The effects of glucagon and insulin on the patient’s PAH activity were investigated. The results are shown in the figure below. In addition, the amount of radioactively-labeled phosphate incorporated into PAH with glucagon treatment was found to be nearly seven-fold greater than in controls.

1. How would you interpret these data?

2. Which hormone activates PAH, and why?

Answer 1

Step 3-1: PCR to amplify the mutated gene

We can use genomic DNA or cDNA from the blood sample. As genomic DNA will provide the whole idea of the type of mutation that is present in the genome. cDNA will tell whether the occurred mutation is a non-sense mutation or not which is resulting in repression or expression of the gene.

Step 3-2: Creating a construct.

Bacterial origin of replication (ORI), Bacterial Ribosome Binding Site (RBS),

Antibiotic resistance gene, Bacterial Terminator sequence, T7 RNA polymerase binding site

As the construct is express in E.coli so we need ORI of bacteria only as with bacterial replication, prokaryotic proteins will only identify bacterial ORI, not the Human ORI. Also for expression of protein we require T7RNA polymerase as it is a highly active polymerase that synthesizes more RNA as compare to the E.coli RNA polymerase, it has a low error rate, Bacterial polymerase inhibitor cant not inhibit this enzyme. RBS should be of bacterial origin, as in translation Bacterial ribosome will bind to bacterial RBS only, and to prevent contamination of any other bacterial growth we will use a resistant gene with our construct.

Step 3-3: Mutation in amino acids

mutations from Leucine to Alanine and Proline to Alanine will most likely to be the cause of the patient's illness. As in these mutations, firstly talking about Leucine to Alanine, the size of leucine(Mw=131Da) is bigger than alanine (Mw=89Da). As leucine is branched amino acid as compare to alanine, this can be responsible for some structural changes that were earlier helping at the active site by providing its bulky R group Considering the second mutation Proline to Alanine, it may cause several changes to folding. As we know in proline there is no free NH3 group, so it mostly makes a cis double bond that provides kink in the structure. Proline may act as a helix breaker for alpha sheets but it stabilizes structures like Beta-turns.

Step 4-1: 30mMNaCl,100mMTrispH7.5

As resins we are using is anion exchanger, so we need to use a pH above the pI of protein which is 6.2. so In the provided options, it's best to use pH= 7.5. As at a pH greater than pI, there will be a negative charge providing elution of protein from the column.

The buffer used should be more in negative charge and more in concentration as compare to our protein attach to the anionic column so that it can strongly displace our protein in one go from the column.

Step 4-2: Was the PAH:-

Expressed = Yes, as all the components that are picked for constructing the construct with our mutated gene are of the bacterial system and we are putting it E.coli which is a prokaryote.

The correct size = Yes, as the mutation will not cause a drastic change in molecular weight.

Soluble = yes, then only it can be obtained through various purification steps.

Bound by the column = no, if it is bound to the column, we can not get in elution buffer and if it will remain bound to column then no further studies could be done on the protein.

Present in the fractions = yes, that's why suitable elution buffer is used.

Step-5: Techniques for amino-acid substitution:-

Computational analysis can be done for each amino acid and even more, suitable mutations can be suggested for the PAH.

RAPD, SNP can be done on the genomic level. it may be too tedious to perform but can help to identify the difference due to single nucleotide differences on high percentage polyacrylamide gel.